Molecular Biology Reports

, Volume 41, Issue 7, pp 4689–4696 | Cite as

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients

  • Bhupesh Singla
  • Anuradha Chakraborti
  • Bal Krishan Sharma
  • Shweta Kapil
  • Yogesh K. ChawlaEmail author
  • Sunil K. Arora
  • Ashim Das
  • Radha K. Dhiman
  • Ajay Duseja


Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.


Hepatitis B virus HBV cccDNA Intrahepatic total HBV DNA Real time PCR Liver biopsy 



The authors have no competing interests. This work was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. (ICMR No: VIR/28/2010-ECD-I). Authors are grateful to ICMR for giving Junior Research Fellowship to Bhupesh Singla (3/1/3JRF-2008/MPD, dated 1/9/2008).

Supplementary material

11033_2014_3339_MOESM1_ESM.tif (281 kb)
Figure S1: Amplification curve for HBV cccDNA quantification standards. (TIFF 280 kb)
11033_2014_3339_MOESM2_ESM.tif (232 kb)
Figure S2: Melting curve for HBV cccDNA quantification standards. (TIFF 232 kb)
11033_2014_3339_MOESM3_ESM.tif (263 kb)
Figure S3: Standard curve made from HBV cccDNA quantification standards. (TIFF 262 kb)


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Bhupesh Singla
    • 1
  • Anuradha Chakraborti
    • 2
  • Bal Krishan Sharma
    • 1
  • Shweta Kapil
    • 1
  • Yogesh K. Chawla
    • 1
    Email author
  • Sunil K. Arora
    • 3
  • Ashim Das
    • 4
  • Radha K. Dhiman
    • 1
  • Ajay Duseja
    • 1
  1. 1.Department of Hepatology, Nehru HospitalPost Graduate Institute of Medical Education and ResearchChandigarhIndia
  2. 2.Department of Experimental Medicine & BiotechnologyPost Graduate Institute of Medical Education and ResearchChandigarhIndia
  3. 3.Department of ImmunopathologyPost Graduate Institute of Medical Education and ResearchChandigarhIndia
  4. 4.Department of HistopathologyPost Graduate Institute of Medical Education and ResearchChandigarhIndia

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